Intro to biological techniques

Question 1

Why is molecular biology useful to us?

Answer 1

Allows us to:

• study genes to elucidate disease mechanisms
• sequence genes to diagnose genetic disorders
• detect infectious pathogens
• synthesise clinically important gene products
• carry out forensic and paternity tests

Question 2

What type of bonds are formed in DNA backbone?

Answer 2

phosphodiester bonds, from 5’ to 3’

Question 3

What gives nucleic acids their overall negative charge?

Answer 3

The phosphate group

Question 4

What 2 things do type II restriction enzymes do?

Answer 4

1. recognise a specific (palindromic) DNA sequence
2. break phosphodiester bonds

Question 5

What are the overhangs called that are sometimes produced?

Answer 5

sticky ends

Question 6

What does DNA ligase do?

Answer 6

Joins the annealed sticky end fragments together by forming new phosphodiester bonds

Question 7

What is an example of vector DNA?

Answer 7

Plasmids, bacteriophages (anything that can be used to transfer DNA into a cell)

Question 8

What is recombinant DNA?

Answer 8

The new combination of bases in the DNA (vector DNA + fragment DNA)

Question 9

What is a good way to separate DNA molecules?

Answer 9

Gel electrophoresis - negative charged dna migrates towards anode (positive) - uses Agarose gel

Question 10

What are the 2 stages of DNA cloning?

Answer 10

1. creating a DNA molecule containting the molecule of interest plus replication signals
2. inserting the DNA into an organism, growing large numbers + then isolating the dna

ideal organisms = small/fastgrowing eg. bacteria, yeast

Question 11

What are characteristics of plasmids making it useful for cloning?

Answer 11

• Mostly circular nucleic acid molecules
• occur naturally in bacteria + yeast
• confer new properties of the host eg. antibiotic resistance
• can replicate independently of bacterial chromosome
• possess OriR signal but require host’s enyzymes for replication

Question 12

Why is E.Coli good for this purpose?

Answer 12

• lab strain of gut flora = harmless
• grows rapidly in simple medium
• easy to introduce plasmid DNA at high efficiency
• easy to extract plasmid DNA

Question 13

Describe the process of cloning

Answer 13

• isolate dna
• isolate region of interest using RE
• ligate into plasmid
• transform into E.Coli
• grow transformed bacteria
• induce protein expression -> purify product
• OR purify plasmid dna -> recover insert using restriction enzyme

Question 14

What is PCR used for?

Answer 14

Exponential amplification of a DNA fragment of a known sequence

Question 15

What are the components used in PCR and why are they?

Answer 15

• template - dna to amplify
• primers - short pieces of ssDNA
• polymerase - thermostable enzyme
• nucleotides - single base mixture
• buffer - to maintain pH
• MgCl2 - essential for polymerase activity

Question 16

Describe step 1/3 of PCR

Answer 16

DENATURATION

Heat + separate DNA strands at 95C.

Question 17

Describe step 2 of PCR

Answer 17

ANNEALING

Primers anneal with template at 50-65C.

Question 18

Describe step 3 of PCR

Answer 18

EXTENSION

DNA polymerase extends strand from primer, 72C.

Question 19

Why is PCR useful?

Answer 19

• no need for laborious cloning process
• requires very little starting material
• highly specific
• can introduce restriction sites at ends
• can be easily scaled up to handle large numbers of samples
• can be carried out in very small vols

Question 20

Describe the 4 steps of the Sickle Cell Test

Answer 20

• Mutation in β globin gene (HBB)

1. Obtain DNA sample
2. PCR amplify region of β globin gene
3. Restriction digest with Ddel
4. Gel electrophoresis of products